Biotin Binding Hardly Affects Electron Transport Efficiency across Streptavidin Solid-State Junctions

The electron transport (ETp) efficiency of solid-state protein-mediated junctions is highly influenced by the presence of electron-rich organic cofactors or transition metal ions. Hence, we chose to investigate an interesting cofactor-free non-redox protein, streptavidin (STV), which has unmatched strong binding affinity for an organic small-molecule ligand, biotin, which lacks any electron-rich features. We describe for the first time meso-scale ETp via electrical junctions of STV monolayers and focus on the question of whether the rate of ETp across both native and thiolated STV monolayers is influenced by ligand binding, a process that we show to cause some structural conformation changes in the STV monolayers. Au nanowire-electrode–protein monolayer–microelectrode junctions, fabricated by modifying an earlier procedure to improve the yields of usable junctions, were employed for ETp measurements. Our results on compactly integrated, dense, uniform, ∼3 nm thick STV monolayers indicate that, notwithstanding the slight structural changes in the STV monolayers upon biotin binding, there is no statistically significant conductance change between the free STV and that bound to biotin. The ETp temperature (T) dependence over the 80–300 K range is very small but with an unusual, slightly negative (metallic-like) dependence toward room temperature. Such dependence can be accounted for by the reversible structural shrinkage of the STV at temperatures below 160 K.

S2 layer (by tapping mode topography), we switched to the contact mode for AFM nanoshaving, using the same AFM tip without changing the sample position. The spring constant of the AFM cantilever was calibrated by deflection sensitivity (α) calculation and thermal-K tuning operation. The exact contact force 1 applied by the AFM cantilever was calculated using the value of deflection sensitivity (reciprocal of the slope obtained from the linear part of the force-distance curve in the repulsive regime) and the thermal-K -derived spring constant (k) of the AFM cantilever:

Contact force [nN] = (Setpoint -Free deflection [volt]) × α [nm/volt] × k[nN/nm]
Nano-shaving is a kind of (high force-induced) contact mode imaging, where the AFM-tip removes surface molecules (proteins in our case) from a much harder underlying substrate.
For the t-STV monolayers, the nano-shaving was performed over ~ 500×500 nm-square scan area with 130-150 nN 1 applied contact force, which was effective for the removal of t-STV/n-STV proteins from the Au-substrate. However, we found that removal of n-STV molecules was relatively difficult, even with higher applied contact force. In order to get a clear topographic view of the nano-shaved area, we switched to the tapping mode after the nanoshaving of the protein layer. The thickness of protein film was obtained from the line-profile at the nano-shaved region of tapping mode AFM image (with 3× 3 µm-square scan area).
Gwyddion-2.58 software was used for processing the AFM images.

Polarization modulation-infrared reflection-absorption spectroscopy (PM-IRRAS)
For PM-IRRAS characterization of our protein monolayers, we used the PEM module of a single-channel Nicolet 6700 spectrometer with a grazing angle accessory and a liquid nitrogen cooled MCT detector. The collected IR spectrum for each sample was the average signal over 2000 scans at 0.8 cm -1 resolution with an incident angle of 80 degree. Dedicated Omnic 8.1 software served for PM-IRRAS data processing. In this work, we focused mainly on the amide-I and amide-II bands and their relative positions. We disentangled the influence of biotin binding on the STV spectrum by deconvoluting the bands, as explained in main text.

Surface potential measurements-Kelvin probe
Kelvin probe (KP)-based surface potential measurements allow for estimating the work function ( ) of a surface, relative to a surface with known (and ambient -stable) reference work function. Our main goal was to check how the different streptavidin surface charge affects the work function of the protein films. We used a macroscopic KP system from S3 Besocke (with gold gauze tip), operated inside a N 2 -filled glovebox at room temperature. For every set of experiments, we first performed the measurement of the contact potential difference (V CPD in volts) between the KP(Au tip) probe and freshly peeled highly oriented pyrolytic graphite (HOPG, the reference) surface. This was followed by the V CPD (in volts) measurement between the KP tip and the surface of protein films. The relative work-function of the protein film was obtained from where the reference has a known value: = 4.6 . 2 Here V CPD (HOPG) and V CPD (Sample) are the contact potential difference between HOPG surface and KP tip and the sample surface relative to KP tip, respectively.

Insights into Au-protein-AuNW junction stability
We found, that the junction (in)stability can often be assessed from the (ir)     its biotin complex (PDB-6J6J) with surface exposed acidic (red) and basic (blue) amino acid residues. Orange circles indicate the position of embedded biotins for two of n-STV subunits (from the top view, as shown) and the white circle represents the surface exposed biotin binding pocket of n-STV after biotin binding. Here 'down' refers to cooling down to 110 K and 'up' refers to heating up to room temperature.
Figure S11: Reversible current-voltage response (as a function of temperature) of the representative junctions (over the bias sweep ± 0.5 V) of t-STV and the t-STV complex (of biotin) monolayers; 'down' means cooling down to 80 K and 'up' means heating up to room temperature.